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Associate

Patricia Moussatche

Education

BA in Biology. Bard College, New York (1998)

PhD in Plant Molecular and Cellular Biology. University of Florida (2004)

Research summary

My current research focuses on understanding the pathway of nucleic acid
incorporation into DNA. By using non-standard bases, we can dissect the
pathway and identify essential enzymes. These non-standard bases can also
be used to create a 6-base genetic code, if we can engineer the enzymes in
the pathway to incorporate them into DNA.

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzymecatalyzed
reactions when neither products nor reactants are spectrophotometrically visible and when
the reaction products are unknown. We report here the use of the multiple injection method of ITC to
characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx),
a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon
dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin
enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx
activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis,
which is equal to the number of moles of product produced times the enthalpy of the reaction (?Happ).
Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are
comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is
coupled to the horseradish peroxidase-catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-
sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to
identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable
to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that
also serve as substrates for the enzyme.

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is
substantially beyond current theoretical and technological capabilities. In pursuit of this
goal, scientists are forced across uncharted territory, where they must answer unscripted
questions and solve unscripted problems, creating new theories and new technologies in
ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery
and paradigm changes in ways that analysis cannot. Described here are the products
that have arisen so far through the pursuit of one grand challenge in synthetic biology:
Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using
genetic and catalytic biopolymers different from those that have been delivered to us by
natural history on Earth. The outcomes in technology include new diagnostic tools that have
helped personalize the care of hundreds of thousands of patients worldwide. In science, the
effort has generated a fundamentally different view of DNA, RNA, and how they work.

Noroviruses are the major cause of global viral gastroenteritis with short incubation times and small inoculums required for infection. This creates a need for a rapid molecular test for norovirus for early diagnosis, in the hope of preventing the spread of the disease. Non-chemists generally use off-the shelf reagents and natural DNA to create such tests, suffering from background noise that comes from adventitious DNA and RNA (collectively xNA) that is abundant in real biological samples, especially feces, a common location for norovirus. Here, we create an assay that combines artificially expanded genetic information systems (AEGIS, which adds nucleotides to the four in standard xNA, pairing orthogonally to A:T and G:C) with loop-mediated isothermal amplification (LAMP) to amplify norovirus RNA at constant temperatures, without the power or instrument requirements of PCR cycling. This assay was then validated using feces contaminated with murine norovirus (MNV). Treating stool samples with ammonia extracts the MNV RNA, which is then amplified in an AEGIS-RT-LAMP where AEGIS segments are incorporated both into an internal LAMP primer and into a molecular beacon stem, the second lowering background signaling noise. This is coupled with RNase H nicking during sample amplification, allowing detection of as few as 10 copies of noroviral RNA in a stool sample, generating a fluorescent signal visible to human eye, all in a closed reaction vessel.

ABSTRACT: Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of
an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1'-β-D-2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1'β-D-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-D-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA.

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.

The steroid hormone progesterone regulates many critical aspects of vertebrate physiology. The nuclear receptor for progesterone functions as a ligand-activated transcription factor, directly regulating gene expression. This type of signalling is referred to as the 'genomic' pathway. Nevertheless, progesterone also stimulates rapid physiological effects that are independent of transcription. This pathway, termed 'non-genomic', is mediated by the mPRs (membrane progesterone receptors). These mPRs belong to a larger class of membrane receptors called PAQRs (progestin and adipoQ receptors), which include receptors for adiponectin in vertebrates and osmotin in fungi. mPRs have been shown to activate inhibitory G-proteins, suggesting that they act as GPCRs (G-protein-coupled receptors). However, PAQRs do not resemble GPCRs with respect to topology or conserved sequence motifs. Instead, they more closely resemble proteins in the alkaline ceramidase family and they may possess enzymatic activity. In the present paper, we highlight the evidence in support of each model and what is currently known for PAQR signal transduction of this non-canonical receptor.

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated.

Proteins belonging to the Progestin and AdipoQ Receptor (PAQR) superfamily of membrane bound receptors are ubiquitously found in fungi. Nearly, all fungi possess two evolutionarily distinct paralogs of PAQR protein, which we have called the PQRA and PQRB subtypes. In the model fungus Saccharomyces cerevisiae, these subtypes are represented by the Izh2p and Izh3p proteins, respectively. S. cerevisiae also possesses two additional PQRA-type receptors called Izh1p and Izh4p that are restricted to other species within the "Saccharomyces complex". Izh2p has been the subject of several recent investigations and is of particular interest because it regulates fungal growth in response to proteins produced by plants and, as such, represents a new paradigm for interspecies communication. We demonstrate that IZH2 and IZH3 gene dosage affects resistance to polyene antifungal drugs. Moreover, we provide additional evidence that Izh2p and Izh3p negatively regulate fungal filamentation. These data suggest that agonists of these receptors might make antifungal therapeutics, either by inhibiting fungal development or by sensitizing fungi to the toxic effects of current antifungal therapies. This is particularly relevant for pathogenic fungi such as Candida glabrata that are closely related to S. cerevisiae and contain the same complement of PAQR receptors.

Bacillus subtilis oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO2 and formate. The enzyme is composed of two cupin domains, each of which contains a Mn(II) ion. Although there is general agreement that Mn(II) in the N-terminal domain mediates OxDC-catalyzed decarboxylation, legitimate questions have been raised concerning the function (if any) of the Mn(II) bound in the C-terminal cupin domain. We have investigated this problem using a series of OxDC mutants in which Mn(II) binding is perturbed by mutagenesis of Glu-101 and Glu-280, which coordinate the metal in the N-terminal and C-terminal domains, respectively. We now demonstrate that decarboxylase activity and total manganese content are sensitive to modifications in either metal-binding glutamate residue. These findings, in combination with EPR measurements, raise the possibility that the C-terminal Mn(II) center can catalyze the decarboxylation reaction. Further support for this conclusion has been provided from a combination of in vivo and in vitro strategies for preparing wild-type OxDC in which Mn(II) is incorporated to a variety of extents. Kinetic characterization of these variants shows that OxDC activity is linearly correlated with manganese content, as might be expected if both sites can catalyze the breakdown of oxalate into formate and CO2. These studies also represent the first unequivocal demonstration that OxDC activity is uniquely mediated by manganese.

Despite more than five decades of extensive studies of thiamin diphosphate (ThDP) enzymes, there remain many uncertainties as to how these enzymes achieve their rate enhancements. Here, we present a clear picture of catalysis for the simple nonoxidative decarboxylase, oxalyl-coenzyme A (CoA) decarboxylase, based on crystallographic snapshots along the catalytic cycled and kinetic data on active site mutants. First, we provide crystallographic evidence that, upon binding of oxalyl-CoA, the C-terminal 13 residues fold over the substrate, aligning the substrate alpha-carbon for attack by the ThDP-C2 atom. The second structure presented shows a covalent reaction intermediate after decarboxylation, interpreted as being nonplanar. Finally, the structure of a product complex is presented. In accordance with mutagenesis data, no side chains of the enzyme are implied to directly participate in proton transfer except the glutamic acid (Glu-56), which promotes formation of the 1',4'-iminopyrimidine tautomer of ThDP needed for activation.